蛋白質體學

1. 蛋白質體學 阮雪芬 Jul 18 & 25, 2003

2. Outline • The characters of proteins • Differences between protein chemistry & proteomics • Why to study proteome • Proteomics • Introduction to proteomics • Definitions of proteomics • The major techniques in current proteomics • Protein-protein interactions

3. The characters of proteins

4. DNA 和蛋白質合成的地方

5. Three Developments Formed the Foundation of the New Biology • The growth of gene, expressed sequence tag (EST), and protein-sequence databases during the 1990s. • The introduction of user-friendly, browser-based bioinformatics tools. • The development of oligonucleotide microarray.

6. Why to study proteome ?

7. Why the Transcriptomic Analyses May Not Have Revealed All Proteins ? • Lack of correlation between transcript and disease-associated protein levels • Translocation of a protein in the disease state rather than simply differential levels of the transcript • Novel/uncharacterized genes that are not highly represented within the "closed system" of a cDNA array

8. Individual proteins Complete sequence analysis Emphasis on structure and function Structural biology Complex mixtures Partial sequence analysis Emphasis on identification by database matching Systems biology Protein chemistryProteomics

9. Introduction To Proteomics

10. Genomics vs. Proteomics Genome “Genomics” DNA mRNA Proteome “Proteomics” Proteins Cell functions

11. Generalized Proteomics Scheme Yarmush & Jayaraman, 2002

12. Definitions of Proteomics

13. Definitions of Proteomics • First coined in1995 • Be defined as the large-scale characterization of theentire proteincomplement of a cell line, tissue, or organism. • Goal: -To obtain a moreglobal and integratedview of biology by studying all the proteins of a cell rather than each one individually.

14. Definitions of Proteomics • The classical definition • Two-dimensional gels of cell lysate and annotation • Two-dimensional gels to visualize differential • protein expression • In the post-genomics era • Protein Identification • Post-translational modifications • Determining Function • Molecular Medicine • Differential display by two-dimensional gels • Protein-Protein Interactions

15. Proteomics Origins • In1975, the introduction of the 2D gel byO’Farrellwho began mapping proteins from E. coli. • The first major technology to emerge for the identification of proteins wasthe sequencing of proteinsby Edman degradation picomole • MS technologyhas replaced Edman degradation to identify proteins femtomole

16. How Proteomics Can Help Drug Development http://www.sciam.com.tw/read/readshow.asp?FDocNo=63&CL=18

17. Why is Proteomics Necessary? • Having complete sequences of genome is not sufficient to elucidate biological function. • A cell is normally dependent upon multitude of metabolic and regulatory pathways for its survival • Modifications of proteins can be determined only by proteomic methodologies • It is necessary to determine the protein expression level • The localization of gene products can be determined experimentally • Protein-protein interactions • Proteins are direct drug targets.

19. Jürgen Drews, 2000

20. Amgen（Applied Molecular Genetics） 成立日期：1980 年4 月8 日 CEO：Kevin W. Sharer 員工人數：6342 市場總值：698.4 億美元 產品項目：重組蛋白藥物 EPOGENR (Epoetin alfa) NEUPOGENR (Filgrastim) INFERGENR (Interferon alfacon-1) 資料來源：彭博資訊社、Zacks.com，6/14/2001

21. 各項產品營業收入 資料來源：Amgen, Inc.

22. The Major Techniques in Current Proteomics

23. The Major Techniques in Current Proteomics • Two-dimensional electrophoresis • IEF strip separation • SDS-PAGE gel separation • Mass Spectrometry • Protein sequencing • Peptide mapping • Others • ICAT • Yeast two hybrid assay • Protein chips

24. Two-dimensional Gel Approach Nature 2000, 405, 837-846

25. Image Matching kDa 150 Increase of 50% 70 Decrease of 50% 60 42 Unmatched spots Matched spots 10 3.5 10 pH

26. www.expasy.ch/ch2d

28. http://www.expasy.ch/melanie/

30. Standard Proteome Analysis by 2DE-MS Mass Fingerprint Searching in http://www.expasych/tools/peptident.html Current Opinion in Chemical Biology 2000, 4:489–494

31. Typical mass spectrometry scheme • peptide mass mapping and tandem mass spectrometry Yarmush & Jayaraman, 2002

32. Ionization State as a Function of pH

33. Two-dimensional Gel Electrophoresis First dimension: IEF (based on isoelectric point) - + Sample acidic basic High MW SDS-PAGE (based on molecular weight) Low MW

34. Staining of Polyacrylamide Gels Silver staining Coomassie blue staining Sypro Ruby staining

35. Image Analysis

36. In-gel Digestion • Enzyme: • trypsin • chymotrypsin

37. * Trypsin * * * * * * Mass Spectrometric Identification of Proteins Mapping Peptide mass fingerprinting (PMF) or peptide mapping

38. 1. Cut protein spot 2. Protein digestion Protease 4. Spot onto MALDI chip 3. Peptide purification 6. Peptide fragment fingerprint 5. MALDI-TOF analysis Protein Identification by MALDI-TOF

39. How Does a Mass Spectrometer Work? Sample input Analyzer Detector Ionization

40. How Does a Mass Spectrometer Work? • Sample Input: • Gas Chromatography (GC), Liquid Chromatography (LC), • Capillary Electrophoresis (CE), Solid crystal etc. • Ionization: • Electrospray, Matrix-assisted Laser Desorption/Ionization (MALDI) etc • Analysis: • quadrupole, time of flight(TOF), ion trap etc. • Detection:

41. Ionization Electrospray

42. Ionization Matrix-Assisted Laser Desorption/Ionization(MALDI) Matrix: - organic acids - benzoic acids

43. Isotope-coded Affinity Tags (ICAT) Linker Biotin Thiol-reactive end group ICAT consists ofa biotin affinity group, a linker regionthat can incorporate heavy or light atoms , anda thiol-reactive end groupfor linkage to cysteines Avidin chromatography

44. A Strategy for Mass Spectrometric Identification of Proteins and Post-translational Modifications NATURE, VOL 405, 15 JUNE 2000

45. Proteome chip ‘proteome chip’composed of 6,566 protein samples representing 5,800 unique proteins, which are spotted in duplicate on a single nickelcoated glass microscope slide39. The immobilized GST fusion proteins were detected using a labeled antibody against GST. (MacBeath G. Nat Genet 2002 Dec;32 Suppl 2:526-32 )

46. Microarrays for Genomics and Proteomics • DNA microarray are used forgenetic analysisas well as expression analysis at themRNAlevel. • Protein microarrays are used for expression analysis at theproteinlevel and in the expansive field ofinteraction analysis.

47. Protein Microarrays In Medical Research • Accelerate immune diagnostics. • The reduction of sample volume----the analysis of multiple tumor markers from a minimun amount of biopsy material. • New possibilities for patient monitoring during disease treatment and therapy will be develpoed based on this emerging technology.

48. Clinical and Biomedical Applications of Proteomics • An approach complementary to genomics is required in clinical situations to better understand epigenetic regulation and get closer to a "holisitic" medical approach. • The potential clinical applications of 2-D PAGE, especially to the analysis ofbody fluidsand tissuebiopsies. • Identifying the origin of body fluid samples or the origin of a tissue biopsy. • Analyzing protein phenotypes and protein post-translational modifications in fluid, cells, or tissues. • Examining the clonality of immunoglobulins and detecting clones which are not seen with conventional techniques. • Monitoring disease processes and protein expression. • Discovering new disease markers and/or patterns in body fluids, cells, or tissues.

49. Body fluids Blood cell Plasma and serum Urine Cerebrospinal fluid Amniotic fluid Synovial fluid Saliva Sweat Tears Semen Solid tissue Heart Brain Thyroid Muscle Malignant diseases Tissue culture Malignant cells Bacterial proteins Clinical applications of 2-D electrophoresis Young & Tracy Journal of Chromatography A, 698 (1995) 163-179

50. Protein-protein Interactions